Polymer-sunitinib conjugates

ABSTRACT

The invention relates to (among other things) polymer-sunitinib conjugates and related compounds. A compound of the invention, when administered by any of a number of administration routes, exhibits advantages over sunitinib in unconjugated form.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2011/067259, filed Dec. 23, 2011, designating theUnited States, which claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application Ser. No. 61/426,919,filed on Dec. 23, 2010, the disclosures of which are incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

This invention comprises (among other things) chemically modified formsof the receptor tyrosine kinase (RTK) inhibitor, sunitinib, which formspossess certain advantages over sunitinib lacking the chemicalmodification. The chemically modified forms of sunitinib describedherein relate to and/or have application(s) in (among others) the fieldsof drug discovery, pharmacotherapy, physiology, organic chemistry andpolymer chemistry.

BACKGROUND OF THE INVENTION

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues withinproteins. Phosphorylation of these hydroxy groups is required for thegrowth, differentiation and proliferation of cells. Thus, virtually allaspects of the cell life cycle depend on normal PK activity. In view ofthe criticality normal PK activity has on healthy cell functioning, itis perhaps not surprising that abnormal PK activity has been related toa host of disorders, ranging from relatively non-life threateningdiseases such as psoriasis to extremely virulent diseases such asglioblastoma (brain cancer).

Among other categorizations, PKs can be divided into two classes, thecytoplasmic protein tyrosine kinases (PTKs) and the transmembranereceptor tyrosine kinases (RTKs). Breifly, the RTKs comprise a family oftransmembrane receptors with diverse biological activity. The HERsubfamily of RTKs includes EGFR (epithelial growth factor receptor),HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylatedligand binding domain, a transmembrane domain and an intracellularcytoplasmic catalytic domain that can phosphorylate tyrosine residues onproteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated alpha subunitsand two beta subunits which cross the cell membrane and which containthe tyrosine kinase domain.

A third RTK subfamily is referred to as the “platelet derived growthfactor receptor” (“PDGF-R”) group, which includes PDGF-R-α, PDGF-R-β,CSFI-R, c-kit and c-fms. These receptors consist of glycosylatedextracellular domains composed of variable numbers of immunoglobin-likeloops and an intracellular domain wherein the tyrosine kinase domain isinterrupted by unrelated amino acid sequences.

Another group, which, because of its similarity to the PDGF-R subfamily(and is sometimes subsumed into the PDGF-R subfamily) is the fetus liverkinase (“flk”) receptor subfamily. This group is believed to be made upof kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1,VEGF-R2), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

Sunitinib is a multi-targeted RTK marketed by Pfizer Inc. under thebrand name SUTENT®. Chemically, sunitinib's systematic name is“N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide”and its chemical formula is provided below.

In vivo, sunitinib inhibits cellular signaling by targeting multipleRTKs, including (VEGFRs) and PDGF-Rs. Both of PDGR-Rs and VEGFRs play arole in tumor proliferation and angiogenesis, and sunitinib's ability toinhibit these targets leads to cancer cell death and reduced tumorvascularization, thereby resulting in tumor shrinkage. Sunitinib alsoinhibits other RTKs, such as KIT, RET, CSF-1R and flt3, therebypotentially making it clinically useful in the treatment of patientssuffering from other cancers.

Treatment with sunitinib, however, is not without drawbacks, includinghand-foot syndrome, stomatitis, and other toxicities.

Therefore, a need exists to provide compounds that can exert the samepharmacology sunitinib has in vivo, yet has an improved side effectprofile. The present invention seeks to address this and/or other needsassociated with administering sunitinib.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a compound is provided, thecompound comprising a sunitinib residue covalently attached via areleasable linkage-containing spacer moiety to a water-soluble,non-peptidic polymer.

In one or more embodiments of the invention, a compound is provided, thecompound having the following structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a compound is provided, thecompound having the following structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a compound is provided, thecompound having the following structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a compound is provided, thecompound having the following structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a composition is provided,the composition comprising (i) a compound comprising a sunitinib residuecovalently attached via a releasable linkage-containing spacer moiety toa water-soluble, non-peptidic polymer, and, optionally, (ii) apharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound as described herein, wherein thecompound is present in a dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicpolymer to sunitinib.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound as described herein to amammal in need thereof.

Additional embodiments of the present conjugates, compositions, methods,and the like will be apparent from the following description, examples,and claims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the phosphate buffer release kinetics of aPEG-sunitinib conjugate of the invention (Compound 4a), as furtherdescribed in Example 1.

FIG. 2 is a plot of the plasma release kinetics of a PEG-sunitinibconjugate of the invention (Compound 4a), as further described inExample 1.

FIG. 3 and FIG. 4 are plots of the mean sunitinib concentration/timecurves for compounds of interests in plasma and tumor, respectively, asfurther described in Example 12.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic polymer” indicates a polymer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” polymer transmits at least 75%, more preferably at least95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-soluble polymeris at least 95% (by weight) soluble in water or completely soluble inwater. With respect to being “non-peptidic,” a polymer is non-peptidicwhen it has less than 35% (by weight) of amino acid residues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer. In the case of a homo-polymer, a single repeating structuralunit forms the polymer. In the case of a co-polymer, two or morestructural units are repeated—either in a pattern or randomly—to formthe polymer. Preferred polymers used in connection with present theinvention are homo-polymers. The water-soluble, non-peptidic polymercomprises one or more monomers serially attached to form a chain ofmonomers. The polymer can be formed from a single monomer type (i.e., ishomo-polymeric) or two or three monomer types (i.e., is co-polymeric).

An “polymer” is a molecule possessing from about 2 to about 2000monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG polymer” or any polyethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though, the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG polymers for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG polymers, thevariable (n) ranges from about 2 to 2000, and the terminal groups andarchitecture of the overall PEG can vary. When PEG further comprises afunctional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer does notresult in formation of an oxygen-oxygen bond (—O—O—, a peroxidelinkage).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. In addition, the end-capping group may contain a targeting moiety.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises a vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell-specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, quantum dots, and the like.

“Branched,” in reference to the geometry or overall structure of apolymer, refers to a polymer having two or more polymers “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of apolymer, refers to a polymer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer branches or forks from a linear structure intoone or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “releasable linkage” is a relatively labile bond that cleaves underphysiological conditions. An exemplary releasable linkage is ahydrolyzable bond that cleaves upon reaction with water (i.e., ishydrolyzed). The tendency of a bond to hydrolyze in water may depend notonly on the general type of linkage connecting two atoms but also on thesubstituents attached to these atoms. Appropriate hydrolyticallyunstable or weak linkages include but are not limited to carboxylateester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides, oligonucleotides, thioesters, andcarbonates. Another exemplary releasable linkage is an enzymaticallyreleasable linkage. An “enzymatically releasable linkage” means alinkage that is subject to cleavage by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, isopropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced. An “alkenyl”group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, acyl, cyano. The substituted alkyl groups may besubstituted once, twice or three times with the same or with differentsubstituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 7 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention causes no significant adverse toxicological effects to apatient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthalenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g., 1-2, 1-3 or 1-4 substituents)chosen from halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a compound described herein that is neededto provide a desired level of active agent and/or conjugate in thebloodstream or in the target tissue. The precise amount may depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of the composition, intended patientpopulation, patient considerations, and may readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a sunitinib residue covalently attachedvia a releasable linkage-containing spacer moiety to a water-soluble,non-peptidic polymer. Following administration to a patient, thereleasable linkage between the sunitinib residue and the water-soluble,non-peptidic polymer cleaves. Depending on the releasable linkage withinthe compound, sunitinib or sunitinib with a relatively small molecularfragment (or “tag”) is released following cleavage.

The sunitinib residue is a residue of the drug sunitinib and refers tothat portion of a polymer-sunitinib conjugate that corresponds tosunitinib.

Exemplary compounds of the invention are encompassed by the followingstructure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

Additional exemplary compounds of the invention are encompassed by thefollowing structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

Additional exemplary compounds of the invention are encompassed by thefollowing structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

Additional exemplary compounds of the invention are encompassed by thefollowing structure:

wherein:

Xr is a releasable linkage-containing spacer moiety; and

POLY is a water-soluble, non-peptidic polymer,

and pharmaceutically acceptable salts thereof.

The Releasable Linkage-Containing Spacer Moiety, “Xr”

In order to effect release of sunitinib (or sunitinib with a relativelysmall molecular fragment), the compounds of the invention include areleasable linkage-containing spacer moiety between the sunitinibresidue and the water-soluble, non-peptidic polymer. Thus, thereleasable linkage must be one that will cleaves in vivo followingadministration to a patient. In this regard, releasable linkages areknown to those of ordinary skill in the art. In addition, whether agiven linkage can serve as a releasable linkage in connection with thecompounds provided herein can be tested through experimentation (e.g.,by administering a compound having the proposed releasable linkage to apatient and testing, e.g., via chromatographic techniques, periodicallyobtained blood samples for indications of cleavage).

Exemplary releasable linkages for use in connection with the compoundsprovided herein include, without limitation, thioether, carbamate,ester, carbonate, urea and enzyme-cleavable peptidic linkages.Thioether, carbamate, ester, carbonate, urea can cleave via aβ-elimination reaction (with or without the enzymatic coordination,e.g., an ester can serve as a releasable linkage herein regardless ofwhether the ester will be cleaved via an esterase). With respect toenzyme-cleavable peptidic linkages, the spacer moiety can include aseries of amino acids known to be a substrate for an enzyme present inthe intended patient population. In this way, upon administration to thepatient, the enzyme-cleavable peptidic linkage-containing compound ofthe invention, will cleave the enzyme-cleavable peptidic linkage viaenzymatic cleavage, thereby releasing sunitinib (or sunitinib with arelatively small molecular fragment). Examples of peptidic linkagessubject to enzymatic cleavage in a given patient population have beendescribed (see, for example, U.S. Patent Application Publication No.2005/0079155) and can be determined experimentally.

In one or more embodiments of the invention, the releasablelinkage-containing spacer moiety, “Xr,” can take the followingstructure:˜[X¹]_(a)—Lr—[X²]_(b)˜  (Formula III)wherein:

(a) is either zero or one;

(b) is either zero or one;

X¹, when present, is a first spacer;

Lr is the releasable linkage; and

X², when present, is a second spacer.

In those instances of Formula III wherein both (a) and (b) are zero, itwill be understood that the releasable linkage-containing spacer is madeup of only the releasable linkage. That is, the releasablelinkage-containing spacer only contains the releasable linkage and noother atoms are present between the sunitinib residue and thewater-soluble, non-peptidic polymer.

In those instances of Formula III wherein either or both of (a) and (b)are one, it will be understood that the releasable linkage-containingspacer contains one or more additional atoms other than those that makeup the releasable linkage. Nonlimiting exemplary spacers (e.g., X¹ andX²) that may flank the releasable linkage include —O—, —NH—, —S—,—C(O)—, —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—,—OC(O)—CH₂—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl. Additionalspacers include, acylamino, acyl, aryloxy, alkylene bridge containingbetween 1 and 5 inclusive carbon atoms, alkylamino, dialkylamino havingabout 2 to 4 inclusive carbon atoms, piperidino, pyrrolidino, N-(loweralkyl)-2-piperidyl, morpholino, 1-piperizinyl, 4-(loweralkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl,4-(methoxy-lower alkyl)-1-piperizinyl, fluorenyl, and guanidine. Forpurposes of the present invention, however, a group of atoms is notconsidered a spacer when it is immediately adjacent to an polymericsegment, and the group of atoms is the same as a monomer of the polymersuch that the group would represent a mere extension of the polymerchain.

When present, a spacer is typically but is not necessarily linear innature. In addition, a spacer is typically but is not necessarilyhydrolytically stable and/or is enzymatically stable. In one or moreembodiments of the invention, the spacer, when present, has a chainlength of less than about 12 atoms (e.g., less than about 10 atoms, lessthan about 8 atoms, and less than about 5 atoms). With respect todetermining length of a particular spacer, length herein is defined asthe number of atoms in a single chain, not counting substituents. Forinstance, a urea linkage such as this,R_(polymer)—NH—(C═O)—NH—R_(drug)′, is considered to have a chain lengthof three atoms (—NH—C(O)—NH—).

The Water-Soluble, Non-Peptidic Polymer, “POLY”

The compounds of the invention include a water-soluble, non-peptidicpolymer. A wide array of polymers can be used and the invention is notlimited with respect to the type (e.g., polyethylene oxide,polyoxazoline, and so forth), size (e.g., from 2 to 4000 monomers insize) and geometry (e.g., linear, branched, multi-armed, and so forth)used.

With respect to type, the water-soluble, non-peptidic polymer can beunderstood as a series of repeating monomers, wherein the type ofmonomer(s) dictates the type of water-soluble, non-peptidic polymer.Exemplary monomers include, but are not limited to the group consistingof: alkylene oxides, such as ethylene oxide or propylene oxide; olefinicalcohols, such as vinyl alcohol, 1-propenol or 2-propenol; vinylpyrrolidone; hydroxyalkyl methacrylamide and hydroxyalkyl methacrylate,where, in each case, alkyl is preferably methyl; α-hydroxy acids, suchas lactic acid or glycolic acid; phosphazene, oxazoline, carbohydratessuch as monosaccharides, alditol such as mannitol; andN-acryloylmorpholine. In one or more embodiments, the water-soluble,non-peptidic polymer is a co-polymer of two monomer types selected fromthis group, or, more preferably, is a homo-polymer of one monomer typeselected from this group. With respect to co-polymers, the two monomertypes in a co-oligomer may be of the same monomer type, for example, twoalkylene oxides, such as ethylene oxide and propylene oxide.

With respect to size, the water-soluble, non-peptidic polymer can be arelatively small or the water-soluble, non-peptidic polymer can berelatively large.

In those embodiments in which a relatively small water-soluble,non-peptidic polymer is present, exemplary values of molecular weightsinclude: below about 2000; below about 1500; below about 1450; belowabout 1400; below about 1350; below about 1300; below about 1250; belowabout 1200; below about 1150; below about 1100; below about 1050; belowabout 1000; below about 950; below about 900; below about 850; belowabout 800; below about 750; below about 700; below about 650; belowabout 600; below about 550; below about 500; below about 450; belowabout 400; below about 350; below about 300; below about 250; belowabout 200; and below about 100 Daltons. Exemplary ranges for arelatively small water-soluble, non-peptidic polymer include from about100 to about 1400 Daltons; from about 100 to about 1200 Daltons; fromabout 100 to about 800 Daltons; from about 100 to about 500 Daltons;from about 100 to about 400 Daltons; from about 200 to about 500Daltons; from about 200 to about 400 Daltons; from about 75 to 1000Daltons; and from about 75 to about 750 Daltons.

For relatively small water-soluble, non-peptidic polymers, the number ofmonomers in will typically fall within one or more of the followingranges: between 1 and about 30 (inclusive); between about 2 and about25; between about 2 and about 20; between about 2 and about 15; betweenabout 2 and about 12; between about 2 and about 10. In certaininstances, the number of monomers in series in the polymer (and thecorresponding conjugate) is one of 1, 2, 3, 4, 5, 6, 7, or 8. Inadditional embodiments, the polymer (and the corresponding conjugate)contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers. Inyet further embodiments, the polymer (and the corresponding conjugate)possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series.Thus, for example, when the water-soluble, non-peptidic polymer includesCH₃—(OCH₂CH₂)_(n)—, “n” is an integer that can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30, and can fall within one or more of the followingranges: between about 1 and about 25; between about 1 and about 20;between about 1 and about 15; between about 1 and about 12; betweenabout 1 and about 10.

When the water-soluble, non-peptidic polymer has 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 monomers, these values correspond to a methoxy end-cappedpoly(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When thepolymer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped poly(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the molecular weight of the water-soluble, non-peptidic polymer inthe compound is relatively large (e.g., greater than 2,000 Daltons), theweight can fall within the range of 2,000 Daltons to about 150,000Daltons. Exemplary ranges, however, include molecular weights in therange of from about 3,000 Daltons to about 120,000 Daltons; in the rangeof from about 5,000 Daltons to about 110,000 Daltons; in the range offrom greater than 5,000 Daltons to about 100,000 Daltons, in the rangeof from about 6,000 Daltons to about 90,000 Daltons, in the range offrom about 10,000 Daltons to about 85,000 Daltons, in the range ofgreater than 10,000 Daltons to about 85,000 Daltons, in the range offrom about 20,000 Daltons to about 85,000 Daltons, in the range of fromabout 53,000 Daltons to about 85,000 Daltons, in the range of from about25,000 Daltons to about 120,000 Daltons, in the range of from about29,000 Daltons to about 120,000 Daltons, in the range of from about35,000 Daltons to about 120,000 Daltons, and in the range of from about40,000 Daltons to about 120,000 Daltons.

Exemplary molecular weights for relatively large water-soluble,non-peptidic polymers include about 2,200 Daltons, about 2,500 Daltons,about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons,about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000Daltons, and about 75,000 Daltons. Branched versions of thewater-soluble, non-peptidic polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) andmulti-arm versions of the water-soluble, non-peptidic polymer (e.g., afour-armed 40,000 Dalton water-soluble polymer comprised of four 10,000Dalton polymers) having a total molecular weight of any of the foregoingcan also be used.

Thus, regardless of whether a relatively small or large water-soluble,non-peptidic polymer is used, when the water-soluble, non-peptidicpolymer is a poly(ethylene oxide), the polymer will comprise a number of(OCH₂CH₂) monomers [or (CH₂CH₂O) monomers, depending on how the PEG isdefined]. As used throughout the description, the number of repeatingunits is identified by the subscript “n” in “(OCH₂CH₂)_(n).” Thus, thevalue of (n) typically falls within one or more of the following ranges:from 2 to about 3400, from about 100 to about 2300, from about 100 toabout 2270, from about 136 to about 2050, from about 225 to about 1930,from about 450 to about 1930, from about 1200 to about 1930, from about568 to about 2727, from about 660 to about 2730, from about 795 to about2730, from about 795 to about 2730, from about 909 to about 2730, andfrom about 1,200 to about 1,900. For any given polymer in which themolecular weight is known, it is possible to determine the number ofrepeating units (i.e., “n”) by dividing the total weight-averagemolecular weight of the polymer by the molecular weight of the repeatingmonomer.

With respect to geometry, any geometry (e.g., linear, branched,multi-armed) can be used in connection with the conjugates of theinvention and the invention is not limited in this regard.

With respect to linear water-soluble, non-peptidic polymers, typically,although not necessarily, a linear water-soluble, non-peptidic polymerwill be terminally end capped with a substantially inert group (e.g.,with a methyl or methoxy group) on the terminus not attached toreleasable linkage-containing spacer moiety. In one or more embodiments,however, compounds of invention having a linear, water-soluble,non-peptidic polymer will not be terminally end capped with asubstantially inert group and will instead have a functional group. Insuch embodiments, the linear, water-soluble, non-peptidic polymer canafford compounds of the invention having two sunitinib residues attachedto it. In another form of such embodiments, the linear, water-soluble,non-peptidic polymer can afford compounds of the invention having asingle sunitinib residue and a residue of a different moiety (e.g., atargeting moiety).

With respect to branched water-soluble, non-peptidic polymers, thesepolymers typically contain a two discernable end capped water-soluble,non-peptidic polymers connected via a branch point, which is connectedthrough a spacer to either a functional group (prior to conjugation) orsunitinib residue. Exemplary branched forms of water-soluble,non-peptidic polymers are described herein and in WO 2005/107815, WO2005/108463, U.S. Pat. Nos. 5,932,462 and 7,026,440, and U.S. PatentApplication Publication No. 2005/0009988. Among other benefits, branchedwater-soluble, non-peptidic polymers—given the presence of twodiscernable water-soluble, non-peptidic polymers—have the potential toprovide greater polymer character compared to, for example, a linearpolymer having a single water-soluble, non-peptidic polymer.

As used herein, reference to a “water-soluble, non-peptidic polymer”(e.g., “POLY”) is considered to include branched and multi-arm formseven though two or more discernable water-soluble, non-peptidic polymerscan be identified.

With respect to multi-arm water-soluble, non-peptidic polymers, thesepolymers typically contain three or more discernable water-soluble,non-peptidic polymers, each having the ability to covalently attach to amoiety of interest, and each typically connected to a central coremoiety (e.g., a residue of a polyol). Among other benefits, multi-armwater-soluble, non-peptidic polymers—given the ability of each arm tocovalently attach to a drug—have the potential to provide greater drugcharacter compared to, for example, a linear polymer having a singledrug attached thereto.

Methods for Synthesizing Compounds of the Invention

The compounds discussed herein can be prepared in a variety of methodsand the invention is not limited in this regard.

In one or more embodiments, the compounds of the prepared by a methodcomprising covalently attaching a water-soluble, non-peptidic polymer tosunitinib. Sunitinib can be obtained commercially as the malate saltfrom Pfizer Inc. In addition, methods for preparing sunitinib aredescribed in U.S. Pat. No. 7,211,600.

With respect to the water-soluble, non-peptidic polymer, such polymerscan be obtained commercially in a form bearing one or more reactivegroups, thereby providing a reagent suited for facile covalentattachment to sunitinib. In this form, the water-soluble, non-peptidicpolymer is sometimes conventionally referred to as a polymeric reagent.Commercial suppliers for polymeric reagents include Sigma-Aldrich (St.Louis, Mo.), Creative PEGWorks (Winston Salem, N.C. USA), SunBioPEG-Shop (SunBio USA, Orinda, Calif.), JenKem Technology USA (Allen,Tex.), and NOF America Corporation (White Plains, N.Y.). Using routineexperimentation, one of ordinary skill in the art can identify polymericreagents having sizes, geometries, and reactive groups and so forth forpreparing the compounds of the invention. For example, it is possible toprepare a series of compounds wherein each member in the series differsin a feature (e.g., the size of the water-soluble, non-peptidic polymer,the type of reactive groups, the ability of a linkage to release, and soforth) and then administer one member in the series to a patientfollowed by periodic detection and quantification (e.g., usingchromatographic techniques) of blood and/or urine samples. Each memberof the series is administered and quantified in a similar way to a naïvepatient. Once each member of the series is tested, the results can bereviewed to determine which feature(s) provided compounds having thedesired effect(s).

Covalently attaching the polymeric reagent to sunitinib is typicallyconducted under conjugation conditions, which conditions includecombining sunitinib with a polymeric reagent (often a molar excess ofpolymeric reagent relative to sunitinib) under conditions oftemperature, pH, time and solvent that allow for covalent attachmentbetween a reactive group of the polymeric reagent to theoxindole-3-methylene or oxindole amide of sunitinib. Additionally,covalent attachment between a reactive group or linker moiety and thepolymeric reagent may be envisioned at the pyrrole amine or thepyrrole-3-carboxamide. For reference, the oxindole-3-methylene, oxindoleamide, pyrrole amine and pyrrole-3-carboxamide of sunitinib areindicated below.

In one or more embodiments, the polymeric reagent used is selected suchthat (i) the reactive group of the polymeric reagent will form acovalent attachment at the oxindole-3-methylene and/or oxindole amide,and (ii) the polymeric reagent includes a releasable linkage (e.g.,prior to being covalently attached) or will include a releasable linkage(e.g., following covalent attachment to sunitinib.

Exemplary conjugation conditions between a given polymeric reagentbearing a reactive group and sunitinib's oxindole-3-methylene, oxindoleamide, pyrrole amine or pyrrole-3-carboxamide will be known to one ofordinary skill in the art based upon the disclosure provided herein andin the context of the relevant literature. See, for example,Poly(ethylene glycol) Chemistry and Biological Applications, AmericanChemical Society, Washington, D.C. (1997).

Exemplary linear polymeric reagents (along with exemplary conjugationconditions for those polymeric reagents) along with the releasablelinkage-containing compounds formed therefrom are presented in Table 1.

TABLE 1 Exemplary Linear Polymeric Reagents and ReleasableLinkage-Containing Compounds Formed Therefrom Exemplary Linear PolymericReagents (and exemplary conjugation conditions) '

  (DMF, TEA) (acetonitrile/H₂O, Na₂CO₃) wherein n is 4 to 2000

  O═C═N—(CH₂CH₂O)₂₋₁₀CH₃ (acetonitrile, DIPEA) wherein n is 4 to 2000

  (tetrahydrofuran/acetonitrile, TEA) wherein n is 4 to 2000

  (acetonitrile, TEA) wherein n is 4 to 2000 and R is amino acidsidechain

  (pH 6.5, DCM) (acetonitrile, TEA) wherein n is 4 to 2000

  wherein n is 4 to 2000

  (acetonitrile, TEA) wherein n is 4 to 2000

  wherein n is 4 to 2000

  (acetonitrile, TEA) wherein n is 100 to 2000

  (acetonitrile/H₂O, Na₂CO₃)

  (acetonitrile, TEA) wherein n is 4 to 2000

  (acetonitrile, TEA) wherein n is 4 to 2000

  (acetonitrile, TEA) wherein n is 4 to 2000 and R is amino acidsidechain Releasable Linkage-Containing Compound Formed Therefrom

  wherein n is 100 to 2000

Exemplary branched polymeric reagents (along with exemplary conjugationconditions for those polymeric reagents) along with the releasablelinkage-containing compounds formed therefrom are presented in Table 2.The branched polymeric reagents set forth in Table 2 can be prepared asdescribed in U.S. Patent Application Publication Nos. 2005/0009988 and2006/0293499.

TABLE 2 Exemplary Linear Polymeric Reagents and ReleasableLinkage-Containing Compounds Formed Therefrom

  (acetonitrile, pyridine) or (methanol/water pH 6-10)

  (acetonitrile, pyridine) or (methanol/water pH 6-10)

  (acetonitrile, pyridine) or (methanol/water pH 6-10)

  (acetonitrile, pyridine) or (methanol/water pH 6-10)

  (acetonitrile, pyridine) or (methanol/water pH 6-10)

  (acetonitrile, TEA) wherein n is 4 to 2000

Exemplary multi-arm versions of compounds of the invention havestructures encompassed by the formula:

wherein:

R is a residue of polyol, polythiol or polyamine bearing at from 3 toabout 50 hydroxyl, thiol or amino groups;

each Q is a linker (and, in one or more embodiments, a hydrolyticallystable linker);

each Xr is a releasable linkage-containing spacer moiety;

each POLY is a water-soluble, non-peptidic polymer; and

q is a positive integer from 3 to about 50 (e.g., 4),

and pharmaceutically acceptable salts thereof.

Additional exemplary multi-arm versions of compounds of the inventionhave structures encompassed by the formula:

wherein:

R is a residue of polyol, polythiol or polyamine bearing at from 3 toabout 50 hydroxyl, thiol or amino groups;

each Q is a linker (and, in one or more embodiments, a hydrolyticallystable linker);

each Xr is a releasable linkage-containing spacer moiety;

each POLY is a water-soluble, non-peptidic polymer; and

q is a positive integer from 3 to about 50 (e.g., 4),

and pharmaceutically acceptable salts thereof.

Additional exemplary multi-arm versions of compounds of the inventionhave structures encompassed by the formula:

wherein:

R is a residue of polyol, polythiol or polyamine bearing at from 3 toabout 50 hydroxyl, thiol or amino groups;

each Q is a linker (and, in one or more embodiments, a hydrolyticallystable linker);

each Xr is a releasable linkage-containing spacer moiety;

each POLY is a water-soluble, non-peptidic polymer; and

q is a positive integer from 3 to about 50 (e.g., 4),

and pharmaceutically acceptable salts thereof.

Additional exemplary multi-arm versions of compounds of the inventionhave structures encompassed by the formula:

wherein:

R is a residue of polyol, polythiol or polyamine bearing at from 3 toabout 50 hydroxyl, thiol or amino groups;

each Q is a linker (and, in one or more embodiments, a hydrolyticallystable linker);

each Xr is a releasable linkage-containing spacer moiety;

each POLY is a water-soluble, non-peptidic polymer; and

q is a positive integer from 3 to about 50 (e.g., 4),

and pharmaceutically acceptable salts thereof.

Due to incomplete conversions, less than 100% yields, and otherunavoidable complications routinely encountered during chemicalsyntheses, exemplary compositions comprising an exemplary “4-arm-PEG”compound can be characterized as compositions comprising four-armcompounds, wherein at least 90% of the four-arm compounds in thecomposition:

(i) have a structure encompassed by the formula,C—[CH₂—O—(CH₂CH₂O)_(n)—CH₂-Term]₄,

wherein

-   -   n, in each instance, is an integer having a value from 5 to 150        (e.g., about 113), and    -   Term, in each instance, is selected from the group consisting of        —OH, —C(O)OH,

(or other activated ester or functional group other than carboxylicacid), and —NH—CH₂—C(O)—O-Sun, wherein Sun is a residue of sunitinib;and

(ii) for each Term in the at least 90% of the four-arm compounds in thecomposition, at least 90% are Sun.

As contemplated by the above structures (i.e., “Formula I-C, multi-arm”,“Formula II-C, multi-arm”, “Formula III-C, multi-arm” and “Formula IV-C,multi-arm”), the compound has “q” number of arms, i.e., from 3 to about50. An exemplary number of arms includes 3, 4, 5, 6, 7, 9, and 10. Inone or more embodiments, the compounds of the invention are preparedfrom multi-armed polymer reagents, which, in turn, are prepared frommulti-arm polymers based on a multi-arm core molecule.

For example, in one approach, a multi-arm polymer can be prepared from amulti-arm core molecule by effectively “growing” a polymer onto eachterminus of a multi-arm core molecule. By way of non-limiting example,it is possible to synthesize a polymer arm onto a polyol (e.g.,pentaerythritol, diglycerol, etc.) via an ethoxylation reaction. Inanother exemplary approach, a multi-arm polymer can be prepared from amulti-arm core molecule by attaching a water-soluble, non-peptidicpolymer onto each terminus of a multi-arm core molecule. The principlesof both approaches are described in the literature and in, for example,U.S. Pat. No. 7,026,440. The invention, however, is not limited withregard to the specific approach taken.

In one or more embodiments, the residue of the polyol, polythiol orpolyamine, “R,” used in connection with the multi-arm polymer is anorganic radical-containing moiety possessing from about 3 to about 150carbon atoms (e.g., from about 3 to about 50 carbon atoms, such as 3, 4,5, 6, 7, 8, 9, and 10). The residue may contain one more heteroatoms(e.g., O, S, or N). In addition, the residue may be linear. In someinstances, the residue may be cyclic.

As previously indicated, the residue of the polyol, polythiol orpolyamine, “R,” that forms the basis of the branching for themulti-armed compounds provided herein, originated from a correspondingpolyol, polythiol or polyamine (prior to be incorporated into themulti-arm structures containing a water-soluble, non-peptidic polymer).In one or more embodiments, the corresponding polyol, polythiol, or apolyamine bears at least three hydroxyl, thiol, or amino groups,respectively, available for polymer attachment. A “polyol” is a moleculecomprising three or more hydroxyl groups. A “polythiol” is a moleculethat comprises three or more thiol groups. A “polyamine” is a moleculecomprising three or more amino groups.

In one or more embodiments, the polyol, polyamine or polythiol willtypically contain 3 to about 25 hydroxyl, or amino groups or thiolgroups, respectively, such as from 3 to about 10 (i.e., 3, 4, 5, 6, 7,8, 9, 10) hydroxyl, amino groups or thiol groups, respectively,preferably from 3 to about 8 (i.e., 3, 4, 5, 6, 7, or 8) hydroxyl, aminogroups or thiol groups, respectively. In one or more embodiments, thenumber of atoms between each hydroxyl, thiol, or amino group will vary,although lengths of from about 1 to about 20 (e.g., from 1 to about 5)intervening atoms, such as carbon atoms, between each hydroxyl, thiol oramino group, are exemplary. In referring to intervening core atoms andlengths, —CH₂— is considered as having a length of one intervening atom,—CH₂CH₂— is considered as having a length of two atoms, and so forth.

Exemplary polyols and polyamines (for which corresponding residues couldbe present in the compounds provided herein) have (Radical)-(OH)_(q) and(Radical)-(NH₂)_(q) structures, respectively, where (Radical)corresponds to an organic-containing radical and q is a positive integerfrom 3 to about 50. Note that in each of Formula I-C, multi-arm, FormulaII-C, multi-arm, Formula III-C, multi-arm and Formula IV-C, multi-arm,the variable “Q,” when taken together with R, typically represents aresidue of the core organic radical as described herein. That is to say,when describing polyols, polythiols and polymer amines, particularly byname, these molecules are being referenced in their form prior toincorporation into a water-soluble polymer-containing structure. So, forexample, a compound of Formula I-C, multi-arm, Formula II-C, multi-arm,Formula III-C, multi-arm and Formula IV-C, multi-arm wherein R is aresidue of the polyol, pentaerythritol [C(CH₂OH)₄], the residue “R”includes carbon (i.e., “C,”) and together with “Q” represents“C(CH₂O—)₄.”

Illustrative polyols include aliphatic polyols having from 1 to 10carbon atoms and from 3 to 10 hydroxyl groups, including for example,trihydroxyalkanes, tetrahydroxyalkanes, polyhydroxy alkyl ethers,polyhydroxyalkyl polyethers, and the like. Cycloaliphatic polyolsinclude straight chained or closed-ring sugars and sugar alcohols, suchas mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol,arabitol, erythritol, adonitol, dulcitol, facose, ribose, arabinose,xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose,mannose, pyranose, altrose, talose, tagitose, pyranosides, sucrose,lactose, maltose, and the like. Additional examples of aliphatic polyolsinclude derivatives of glucose, ribose, mannose, galactose, and relatedstereoisomers. Aromatic polyols may also be used, such as1,1,1-tris(4′-hydroxyphenyl)alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, 2,6-bis(hydroxyalkyl)cresols, and thelike. Other core polyols that may be used include polyhydroxycrownethers, cyclodextrins, dextrins and other carbohydrates (e.g.,monosaccharides, oligosaccharides, and polysaccharides, starches andamylase).

Exemplary polyols include glycerol, trimethylolpropane, pentaerythritol,dipentaerythritol, tripentaerythritol, ethoxylated forms of glycerol,trimethylolpropane, pentaerythritol, dipentaerythritol,tripentaerythritol. Also, preferred are reducing sugars such as sorbitoland glycerol oligomers, such as diglycerol, triglycerol, hexaglyceroland the like. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.Additionally, a polyglycerol having an average of 24 hydroxyl groups isalso included as an exemplary polyol.

Exemplary polyamines include aliphatic polyamines such as diethylenetriamine, N,N′,N″-trimethyldiethylene triamine, pentamethyl diethylenetriamine, triethylene tetramine, tetraethylene pentamine, pentaethylenehexamine, dipropylene triamine, tripropylene tetramine,bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, andN,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines thatcan be used in the present invention include putrescine, spermidine, andspermine. Numerous suitable pentamines, tetramines, oligoamines, andpentamidine analogs suitable for use in the present invention aredescribed in Bacchi et al. (2002) Antimicrobial Agents and Chemotherapy,46(1):55-61, which is incorporated by reference herein.

Provided below are illustrative structures corresponding to residues ofpolyols [although each structure is depicted with the oxygen atom (“O”)derived from the corresponding hydroxyl group, each “O” can besubstituted with sulfur (“S”) or NH to depict the corresponding residueof a polythiol or polyamine, respectively). Note that the residues shownbelow would be understood in terms of compounds of: Formula I-C,multi-arm; Formula II-C, multi-arm; Formula III-C, multi-arm; andFormula IV-C, multi-arm, as corresponding to “R” and “Q.” In any event,conjugates based on any of the illustrative structures set forth beloware included as part of the invention.

wherein m is a positive integer from 0-40 [e.g., 0-10, for example, 0-5(i.e., 0, 1, 2, 3, 4, 5)].

Water-soluble, non-peptidic-containing multi-arm polymers (used as, forexample, multi-arm polymeric reagents to prepare compounds of theinvention) based on the above-described polyols, polythiols andpolyamines and others are described in WO 2007/098466, WO 2010/019233and U.S. Pat. No. 7,744,861. These references and others describemethods for preparing such multi-arm polymers.

The linker, Q, serves to connect the residue of the polyol, polythiol orpolyamine bearing at from 3 to about 50 hydroxyl, thiol or amino groups,“R,” to each water-soluble, non-peptidic polymer, POLY, in compoundsaccording to: Formula I-C, multi-arm; Formula II-C, multiarm; FormulaIII-C, multi-arm; and Formula IV-C, multi-arm. In this regard, theinvention is not limited with respect to the specific linker used. Inone or more embodiments, the linker between the residue, “R,” and thewater-soluble, non-peptidic polymer, POLY, is a hydrolytically stablelinker).

In one or more embodiments of the invention, the linker, Q, isinfluenced by the approach used to form the multi-arm polymer employedin preparing the compounds of the invention. For example, if awater-soluble, non-peptidic polymer bearing a functional group reactiveto a hydroxyl, thiol or amine is reacted with a polyol, polythiol orpolyamine, respectively, the linker, Q, may include one or more atomsincorporating the bond formed between the termini of the polyol,polythiol or polamine and the beginning of the repeating monomers of thewater-soluble, non-peptidic polymer, POLY. Illustrative linkingchemistries in this regard (along with the resulting linkers) aredescribed in the literature and in, for example, Wong (1991) “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton, Fla.,and Brinkley (1992) Bioconjug. Chem. 3:2013.

In one or more embodiments of compounds of: Formula I-C, multi-arm;Formula II-C, multi-arm; Formula III-C, multi-arm; and Formula IV-C,multi-arm, Q contains at least one heteratom such as O, or S, or NH,where the atom proximal to R in Q, when taken together with R, typicallyrepresents a residue of an organic radical-containing core of thepolyol, polythiol or polyamine. Generally, the linker, Q, contains from1 to about 10 atoms (e.g., from 1 to about 5 atoms). The linker, Q,typically contains a number of atoms selected from the group consistingof: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Illustrative Qs include O, S,—NH—, —NH—C(O)— and —C(O)—NH—.

The remaining variables in each of: Formula I-C, multi-arm; FormulaII-C, multi-arm; Formula III-C, multi-arm; and Formula IV-C, multi-arm,include the water-soluble, non-peptidic polymer, POLY and the releasablelinkage-containing spacer moiety, both of which have already beendiscussed. With respect to compounds encompassed by: Formula I-C,multi-arm; Formula II-C multi-arm; Formula III-C, multi-arm; and FormulaIV-C, multi-arm, however, typical molecular weights for thewater-soluble, non-peptidic polymer (e.g., each POLY) include: about200, about 250, about 300, about 400, about 500, about 600, about 700,about 800, about 900, about 1,000, about 1,500, about 2,000, about3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 7,500,about 8,000, about 9,000, about 10,000, about 12,000, about 15,000,about 17,500, about 18,000, about 19,000 and about 20,000 Daltons.

Utility and Testing of Compounds

Animal models (rodents and dogs) can used to study oral drug transport.In addition, non-in vivo methods include rodent everted gut excisedtissue and Caco-2 cell monolayer tissue-culture models. These models areuseful in predicting oral drug bioavailability (thereby providing anindication of whether a given compound of the invention can beadministered orally).

To test for binding activity, a compound can be tested using in vitrobinding studies to receptors using various cell lines expressing thesereceptors. In vitro binding studies known to those of ordinary skill inthe art can be used to test the binding for a receptor of interest.

The following assay may be used to determine the level of activity andeffect of a compound on protein kinases. The assay is performed in anELISA (Enzyme-Linked Immunosorbent Sandwich Assay) format [Voller et al.(1980), “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371]. The general procedure is as follows:

a compound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound. Similar assays can bedesigned along the same lines for any protein kinase using techniqueswell known in the art.

Using this basic approach, one can test over 80 protein kinases,including GST-Flk1, pyk2, PYK2, FGFR-1R, EGFR, PDGFR, HER-2, CDK2, andIGF-1.

The compounds of the invention may be tested in animal models of cancersto determine their cancer-inhibition potential. An exemplary model isthe xenograft-based assay. In this assay, the ability of human tumors togrow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides auseful in vivo model for studying the biological response to therapiesfor human tumors. Since the first successful xenotransplantation ofhuman tumors into athymic mice [Rygaard et al. (1969) Acta Pathol.Microbial. Scand. 77:758-760], many different human tumor cell lines(e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck,glioblastoma, bone, and malignant melanomas) have been transplanted andsuccessfully grown in nude mice.

In addition to an approach as provided in the Experimental, thefollowing assays may be used to determine the level of activity,specificity and effect of the different compounds of the presentinvention. Three general types of assays are useful for evaluatingcompounds: cellular/catalytic, cellular/biological and in vivo. Theobject of the cellular/catalytic assays is to determine the effect of acompound on the ability of a tyrosine kinase to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a tyrosine kinase in a cell. The object of the invivo assays is to determine the effect of a compound in an animal modelof a particular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC #CCL 107), A375 cells (melanoma, ATCC #CRL 1619),A431 cells (epidermoid carcinoma, ATCC #CRL 1555), Calu 6 cells (lung,ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL 1435), SKOV3TP5 cells andNIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR,IGF-1R or any other test kinase. The following protocol can be used toperform xenograft experiments.

Female athymic mice (BALB/c, nu/nu) are maintained under clean-roomconditions in micro-isolator cages with Alpha-dri bedding. They receivesterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium [for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)]. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for ten minutes. Pellets are resuspended in sterilePBS or media (without FBS) to a particular concentration and the cellsare implanted into the hindflank of the mice (8-10 mice per group,2-10×10⁶ cells/animal). Tumor growth is measured over 3 to 6 weeks usingvenier calipers. Tumor volumes are calculated as a product oflength×width×height. P values are calculated using the Students t-test.Test compounds in 50-100 μL excipient (DMSO, or VPD:D5W) can bedelivered by IP injection at different concentrations generally startingat day one after implantation.

The compounds of the invention may be administered per se or in the formof a pharmaceutically acceptable salt, and any reference to thecompounds of the invention herein is intended to includepharmaceutically acceptable salts. If used, a salt of a compound asdescribed herein should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Such pharmacologically and pharmaceutically acceptable saltscan be prepared by reaction of the compound with an organic or inorganicacid, using standard methods detailed in the literature. Examples ofuseful salts include, but are not limited to, those prepared from thefollowing acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulfonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

The compounds of the invention may contain one or more chiral centersand for each chiral center, the invention contemplates each opticalisomer as well as any combination or ratio of or an optically activeform, for example, a single optically active enantiomer, or anycombination or ratio of enantiomers (e.g., scalemic and racemicmixtures). In addition, the small molecule drug may possess one or moregeometric isomers. With respect to geometric isomers, a composition cancomprise a single geometric isomer or a mixture of two or more geometricisomers.

The present invention also includes pharmaceutical preparationscomprising a compound as provided herein in combination with apharmaceutical excipient. Generally, the compound itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol,myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines, fatty acidsand fatty esters; steroids, such as cholesterol; and chelating agents,such as EDTA, zinc and other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. The optimal amount of any individual excipient isdetermined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (as alyophilizate or precipitate, which can be in the form of a powder orcake), as well as formulations prepared for injection, which are liquidand require the step of reconstituting the dry form of parenteralformulation. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions,normally being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The compounds of the invention can also be administered through the skinusing conventional transdermal patch or other transdermal deliverysystem, wherein the conjugate is contained within a laminated structurethat serves as a drug delivery device to be affixed to the skin. In sucha structure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The compounds of the invention can also be formulated into a suppositoryfor rectal administration. With respect to suppositories, the compoundis mixed with a suppository base material which is (e.g., an excipientthat remains solid at room temperature but softens, melts or dissolvesat body temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the compound (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

In some embodiments of the invention, the compositions comprising thecompounds of the invention may further be incorporated into a suitabledelivery vehicle. Such delivery vehicles may provide controlled and/orcontinuous release of the compounds and may also serve as a targetingmoiety. Non-limiting examples of delivery vehicles include, adjuvants,synthetic adjuvants, microcapsules, microparticles, liposomes, and yeastcell wall particles. Yeast cells walls may be variously processed toselectively remove protein component, glucan, or mannan layers, and arereferred to as whole glucan particles (WGP), yeast beta-glucan mannanparticles (YGMP), yeast glucan particles (YGP), Rhodotorula yeast cellparticles (YCP). Yeast cells such as S. cerevisiae and Rhodotorulaspecies are preferred; however, any yeast cell may be used. These yeastcells exhibit different properties in terms of hydrodynamic volume andalso differ in the target organ where they may release their contents.The methods of manufacture and characterization of these particles aredescribed in U.S. Pat. Nos. 5,741,495, 4,810,646, 4,992,540, 5,028,703,5,607,677 and U.S. Patent Application Publication Nos. 2005/0281781 and2008/0044438.

The invention also provides a method for administering a compound of theinvention as provided herein to a patient suffering from a conditionthat is responsive to treatment with the compound. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe compound (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

In one or more embodiments of the invention, a method is provided, themethod being directed to a method of treating diseases mediated byabnormal protein kinase activity, in particular, receptor tyrosinekinases (RTKs), non-receptor protein tyrosine kinases (CTKs) andserine/threonine protein kinases (STKs), in a patient, in particularhumans, which method comprises administering to said patient apharmaceutical composition comprising a compound of the invention asdescribed herein. Such diseases include, by way of example and notlimitation, cancer, diabetes, hepatic cirrhosis, cardiovascular diseasesuch as atherosclerosis, angiogenesis, immunological disease such asautoimmune disease and renal disease.

In one or more embodiments of the invention, the invention is directedto the use of a compound of the invention as described herein in thepreparation of a medicament which is useful in the treatment of adisease mediated by abnormal PK activity.

The actual dose to be administered will vary depend upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 1000mg, preferably in doses from 0.01 mg/day to 750 mg/day, and morepreferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given compound of the invention (again,preferably provided as part of a pharmaceutical preparation) can beadministered in a variety of dosing schedules depending on the judgmentof the clinician, needs of the patient, and so forth. The specificdosing schedule will be known by those of ordinary skill in the art orcan be determined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Once the clinical endpoint has beenachieved, dosing of the composition is halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All non-PEG chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofwater-soluble polymer reagents can be prepared using art-knowntechniques described in the literature unless otherwise indicated.

¹H NMR (nuclear magnetic resonance) data was generated by an NMRspectrometer. A list of certain compounds as well as the source of thecompounds is provided below.

Example 1 Synthesis of Amino-Diethyleneglycol Linked PEG-SunitinibConjugates

This example references one or more of the following compounds.

Synthesis of(Z)-2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamicchloride (Compound 1)

In a 500 mL round-bottomed flask was suspended sunitinib (2.0 g, 5.1mmol) in THF (200 mL). To this yellow suspension was added triethylamine(11.8 mL, 84 mmol). The suspension was heated in an oil bath withstirring at 60° C. for several minutes to give an orange solution. Thesolution was cooled for several minutes before transfer to thetriphosgene reaction.

(Caution: To prevent release of toxic phosgene gas from either thereaction apparatus or rotary evaporator, the equipment setups weresparged through a sodium hydroxide scrub solution via an over pressureor exhaust port.) In a separate 1 L round-bottomed flask was addedtriphosgene (1.6 g, 5.4 mmol) in THF (40 mL) to give a colorlesssolution. A sunitinib-TEA solution was transferred into this triphosgenesolution. The reaction mixture rapidly formed a yellow-orangesuspension. After approximately one hour, the solvent was evaporatedunder reduced pressure. The crude solids were slurried with anhydrousTHF (120 mL) and solvent was evaporated. The crude product was thenplaced under high vacuum for several hours. Crude Yield: 5.2 g of orangesolid. HPLC analysis was on a C18 silica column applying an acetonitrilegradient with 0.1% TFA; retention times observed were sunitinib 4.4minutes and carbamoyl chloride product 6.0 minutes with 94% substitutionat 370 nm. The carbamoyl chloride product was further characterized byreaction with excess n-butylamine to form(Z)—N-(butylcarbamoyl)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamideand analyzed by LC-MS ([C₂₇H₃₇FN₅O₃]⁺ expected M+H=498.29. foundM+H=498.3).

Synthesis of tert-butyl 2-(2-hydroxyethoxy)ethylcarbamate (Compound a)

In a 1 L round-bottomed flask was dissolved 2-(2-aminoethoxy)ethanol (5mL, 50 mmol) in Dioxane (100 mL) to give a colorless solution.Di-tert-butyl dicarbonate (16.4 g, 75 mmol) was added. The reactionmixture was diluted with 1 M sodium hydroxide (200 mL). Precipitation ofwhite solid was observed, after several hours stirring. The reactionmixture was diluted with water (˜200 mL) to dissolve the precipitatedsolids. After 48 hours, the reaction solution was extracted two timeswith hexanes (25 mL, 13 mL). The aqueous/dioxane layer was adjusted toacidic pH with HCl (6 M then 1 M) and was extracted two times with DCM(200 mL, 100 mL). The combined DCM layers were washed with brine/diluteHCl (˜300 mL) and then brine (˜300 mL). The organic layer was dried oversodium sulfate (50 g), filtered and evaporated at reduced pressure. TLCconditions were HOAc/MeOH/DCM 1:2:7 and product R_(f) 0.9 was visualizedby ninhydrin stain. No evidence for starting amine R_(f) 0.1 or otherninhydrin positive impurities were shown by the TLC. ¹H-NHR (d₆-DMSO): δ(ppm) 1.4 (9H, s, CH₃); 3.1 (2H, m, CH₂); 3.4 (4H, m, CH₂); 3.5 (2H, m,CH₂); 4.6 (1H, t, OH); 6.8 (1H, s, NH). Exchangeable protons wereevaluated by the addition of H₂O to the NMR sample in d₆-DMSO and nearlysimilar integration for δ (ppm) 4.6 (OH) and 6.8 (NH [Boc]).

Method A:

Synthesis of (Z)-(tert-butyl 2-(2-hydroxyethoxy)ethylcarbamate)2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1a)

In a 50 mL flask was crude Compound 1 (4.8 g, 4.7 mmol) in tert-butyl2-(2-hydroxyethoxy)ethylcarbamate (Compound a) (24 mL, 120 mmol) to givea orange suspension. The reaction mixture was heated at 50° C. in an oilbath and triethylamine (1.3 mL, 9.5 mmol) was added. The reaction wasremoved from heat after 40 minutes. After 1-2 hours at room temperature,the reaction was diluted with MeOH (120 mL) and then precipitated with100 mM phosphate pH 7.5 (2.2 L). The suspension was stirred on ice for30 minutes before it was filtered to collect orange solids. Thefiltercake was washed with cold 100 mM phosphate pH 7.5 (50 mL). Thecrude product was dissolved with DCM (100 mL, 50 mL) and washed withbrine solution (100 mL), dried over sodium sulfate (15 g) and filtered.The filtrate was evaporated at reduced pressure. Crude yield was 2.7 gof a red-orange solid. The crude product was purified further on aBiotage Flash silica column with a DCM/MeOH gradient program. Productfractions were combined and evaporated at reduced pressure. Yield was1.55 g of yellow powder. HPLC analysis was on a C18 silica columnapplying an acetonitrile gradient with 0.1% TFA; retention timesobserved were sunitinib 3.5 minutes and product 8.9 minutes with 97%purity at 370 nm. Analysis by LC-MS ([C₃₂H₄₅FN₅O₇]⁺ expected M+H=630.33.found M+H=630.4). ¹H-NMR (d₆-DMSO): δ (ppm) 0.9 (6H, t, CH₃); 1.3 (9H,s, CH₃); 2.3 (3H, s, CH₃); 2.4 (3H, s, CH₃); 2.5 (˜4H, m, CH₂); 2.6 (2H,t, CH₂); 3.0 (2H, m, CH₂); 3.3 (2H, t, CH₂); 3.4 (2H, m, CH₂); 3.8 (2H,t, CH₂); 4.1 (2H, m, CH₂); 6.7 (˜1H, t, NH [Boc]); 6.8 (1H, m, Ar); 7.0(1H, m, Ar); 7.7 (1H, s, CH); 7.8 (1H, d, Ar); 10.9 (˜1H, s, NH[oxindole]); 13.8 (1H, s, NH [pyrrole]). ¹³C-NMR (d₆-DMSO): δ (ppm) 10.2(CH₃); 11.6 (2C, CH₃); 12.8 (CH₃); 28.2 (3C, CH₃); ˜39.5 (CH₂); 43.0(CH₂); 46.5 (2C, CH₂); 50.8 (CH₂); 65.6 (CH₂); 67.8 (CH₂); 69.1 (CH₂);77.5 (C); 105.9, 106.1 (d, Ar); 110.0, 110.1 (d, Ar); 112.5, 112.7 (d,Ar); 115.4 (Ar); 120.9 (pyrrole); 124.7 (CH); 125.9 (pyrrole); 127.0,127.0 (d, C); 130.6 (pyrrole); 134.7 (Ar); 137.4 (pyrrole); 154.5(C(O)); 155.5 (C(O)); 157.3, 159.2 (d, Ar); 167.4 (C(O)); 169.6 (C(O)).Characterization was supported by 2D-NMR experiments including:¹H-¹H-COSY, ¹H-¹³C-HSQC, and ¹H-¹³C-HMBC. Exchangeable protons wereevaluated by the addition of H₂O to the NMR sample in d₆-DMSO whichdemonstrated loss of integration for δ (ppm) 10.9 (NH [oxindole]),significantly diminished integration for δ (ppm) 13.8 (NH [pyrrole]) andslightly lower integration for δ (ppm) 6.7 (NH [Boc]).

Method B:

Synthesis of (Z)-2-(2-aminoethoxy)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamatetrihydrochloride (Compound 2a)

In a 250 mL round-bottomed flask was dissolved Compound 1a (1.5 g, 2.4mmol) in dioxane (13 mL) to give a orange solution. A solution ofdioxane 4 M HCl (88 mL) was added. Precipitation of red-orange solid wasobserved. After one hour the reaction solvent was evaporated at reducedpressure. The solids were dissolved in MeOH (60 mL) and then solvent wasevaporated at reduced pressure. MeOH dissolution and evaporation wasrepeated. Crude yield 1.52 g orange solid. HPLC analysis was on a C18silica column applying an acetonitrile gradient with 0.1% TFA; retentiontimes observed were sunitinib 3.6 minutes and product 2.2 minutes with96% purity at 370 nm. Analysis by LC-MS ([C₂₇H₃₇FN₅O₅]⁺ expectedM+H=530.28. found M+H=530.3).

Method C:

Synthesis of (Z)-2-(2-(3-(mPEG 20,000)propanamido)ethoxy)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 3a)

In a 13 mL test tube was added Compound 2a (10.9 mg, 0.02 mmol).Followed by a mixture of acetonitrile (1 mL), triethylamine (10 μL)) andDMF (0.3 mL) and then addition of mPEG-SPA 20K (0.3 g, 0.015 mmol).After 24 hours, a portion of the solvent was evaporated under nitrogenstream. The solution was heated at 45° C. and slowly diluted withanhydrous isopropanol (12 mL). The solution was removed from the heatand was slowly cooled to 10-15° C. The resulting slurry was filtered andwashed with additional anhydrous IPA. Residual solvent was evaporated atreduced pressure. Yield 0.25 g yellow powder. HPLC analysis was on a C18silica column applying an acetonitrile gradient with 0.1% TFA; retentiontimes observed were sunitinib 3.5 minutes and product 10.8 minuteswith >99% purity at 370 nm. ¹H-NHR (CDCl₃): δ (ppm) 1.5 (˜6H, m, CH₃);2.3 (3H, s, CH₃); 2.4 (˜3H, s, CH₃); 3.2 (˜3H, s, OCH₃); 3.6 (˜1800H,bs, PEG backbone); 4.3 (˜4H, m, CH₂); 6.8-7.0 (˜3H, bm, Ar, NH); 7.2(˜1H, m, Ar); 7.3 (˜6H, s, CH); 8.7 (˜1H, s, NH); 12.5 (˜0.5H, s, COOH);data above 12.8 ppm not available. Substitution ˜66% by NMR analysis.

Method D:

Synthesis of (Z)-2-(2-(2-(4-armPEG 20,000)acetamido)ethoxy)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4a)

To a 500 mL round bottom flask was added Compound 2a (1.27 g, 2.0 mmol).The solid was dissolved in acetonitrile (22 mL) and DMF (10 mL).4armPEG_(20k)-SCM (8.2 g, 0.014 mmol) was added. After the PEGdissolved, triethylamine (1.34 mL, 9.6 mmol) was added. After threehours, the solvent was evaporated under reduced pressure to provide athick oil. The product was precipitated by slow addition of anhydrousIPA (300 mL). The solid was washed three times with anhydrous IPA (220mL×3) and diethyl ether (200 mL). Residual solvent was evaporated atreduced pressure. Yield was 8.1 g yellow powder. HPLC analysis was on aC18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.5 minutes and product 10.8minutes with >99% purity at 370 nm. ¹H-NHR (CDCl₃): δ (ppm) 1.4 (24H,bs, CH₃); 2.4 (24H, ds, CH₃); 3.3 (˜16H, bm, CH₂); 3.6 (˜1800H, bm, PEGbackbone); 3.9 (8H, s, CH₂); 4.2 (˜8H, bm, CH₂); 6.9 (8H, m, Ar); 7.2(8H, m, Ar, NH); 8.9 (4H, s, NH); data above ˜12.8 ppm not available.Substitution 89% by NMR analysis.

Method E:

In vitro release for conjugates in buffer: In separate containersCompound 2a (˜0.1-0.5 mg/mL), Compound 3a (˜0.5-5 mg/mL) and Compound 4a(˜0.2-1 mg/mL) were dissolved in 100 mM phosphate buffer pH 7.5 (orother pH values as indicated). The conjugates were filtered through a0.2 micron filter into a HPLC vial. The HPLC sample vials were incubatedat 37° C. and injected at various intervals on a HPLC system with C18silica column applying an acetonitrile gradient with 0.1% TFA; retentiontimes observed were sunitinib ˜3.5 minutes and conjugates either ˜2.2minutes for Compound 2a or ˜10.8 minutes for Compounds 3a or 4a. Releasedata for the t_(1/2) values were obtained from the slope of the linearfit to a plot of ln(A_((conjugate))/A_(0(conjugate))) vs. time,according to the first order rate law. A plot of the results forCompound 4a is provided in FIG. 1.

In vitro release for conjugate in plasma: PEG-sunitinib conjugates(e.g., Compound 4a) were provided as powders and on the day of theexperiment dissolved in water to obtain a stock solution of 2.0 mg/mLTest System (6-22% loading). All plasma samples were obtained fromBioreclamation (Hicksville, N.Y.) or equivalent, sodium heparinized, andstored at −80° C. until use. Plasma evaluated for these experiments werepooled male Sprague-Dawley rat plasma and pooled male Beagle dog plasma.A 10 mg/mL PMSF solution in DMSO containing 1% acetic acid=PMSF/DMSO wasprepared. PEG-sunitinib conjugate (2.0 mg/mL) stock solutions in waterwere prepared. Plasma was prewarmed in a shaking water bath at 37° C.for 15 minutes prior to each experiment. An appropriate volume of thePEG-sunitinib conjugate stock solution was added to 7.0 mL plasma (TBD,30% Phosphate or in CO₂ chamber) to obtain a final PEG-sunitinibconjugate concentration of ˜5 μg/mL (sunitinib equivalents, based on aloading factor of 6-22%). Samples were prepared and in triplicate (n=3)for each matrix. Samples were incubated in a shaking water bath at 37°C. Aliquots (250 μL solution at each timepoint) were removed from eachincubation tube at t=0, 15, 30, 60 minutes, 2, 4, 6, 8, 12, and 24hours, respectively and combined with 10 μL of 10 mg/mL PMSF/DMSO and 1%glacial acetic acid in pre-cooled tubes. The samples were split into twoaliquots (125 μL each). Aliquots were immediately flash frozen using dryice and stored at −80° C. until analysis. CC and QC standards ofsunitinib and PEG-sunitinib conjugate were prepared in respective plasmamatrix already treated with PMSF and AA. Aliquots were thawed andanalyzed on HPLC-MS against calibration curve to determineconcentrations of both PEG-sunitinib and sunitinib. Release data for thet_(1/2) values were estimated from the slope of the linear fit to a plotof ln([conjugate]) vs. time, according to the first order rate law. Aplot of the results for Compound 4a is provided in FIG. 2.

Example 2 Synthesis of Amino-Propanol Linked PEG-Sunitinib Conjugates

Synthesis of (Z)-(tert-butyl 3-hydroxypropylcarbamate)2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1b)

Synthesis was conducted as described in Method A substituting tert-butyl3-hydroxypropylcarbamate (Compound b) (0.83 mL, 4.8 mmol) to give aorange suspension. Product precipitation in phosphate buffer andextraction were omitted. Purified product yield was 41 mg of yellowpowder. HPLC analysis was on a C18 silica column applying anacetonitrile gradient with 0.1% TFA; retention times observed weresunitinib 3.7 minutes and product 9.1 minutes with 99% purity at 370 nm.Analysis by LC-MS ([C₃₁H₄₃FN₅O₆]⁺ expected M+H=600.32. found M+H=600.3).¹H-NHR (d₆-DMSO): δ (ppm) 0.9 (6H, bm, CH₃); 1.3 (9H, s, CH₃); 1.5 (2H,m, CH₂); 2.3 (3H, s, CH₃); 2.4 (3H, s, CH₃); 2.6 (2H, bm, CH₂); 2.8 (2H,m, CH₂); 3.8 (2H, bs, CH₂); 4.0 (2H, t, CH₂); 6.8 (1H, t, NH); 6.9 (1H,m, Ar); 7.0 (1H, m, Ar); 7.7 (1H, s, CH); 7.8 (1H, m, Ar); 10.9 (1H, s,NH); data above ˜12.8 ppm not available.

Synthesis of (Z)-3-aminopropyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamatetrihydrochloride (Compound 2b)

Synthesis was conducted as described in Method B substituting Compound1b (34 mg, 0.06 mmol). Crude yield 65 mg orange solid. HPLC analysis wason a C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.7 minutes and product 2.7minutes with 99% purity at 370 nm. Analysis by LC-MS ([C₂₆H₃₅FN₅O₄,]⁺expected M+H=500.27. found M+H=500.3).

Synthesis of (Z)-3-(3-(mPEG 20,000)propanamido)propyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-pyrrole-3-carbonyl)carbamate(Compound 3b)

Synthesis was conducted as described in Method C substituting Compound2b (3.6 mg, 0.006 mmol). Yield 77 mg yellow powder. HPLC analysis was ona C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.7 minutes and product 10.8minutes with 97% purity at 370 nm.

Synthesis of (Z)-3-(2-(4-armPEG 20,000)acetamido)propyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4b)

Synthesis was conducted as described in Method D substituting Compound2b (14 mg, 0.023 mmol). Yield was 70 mg yellow powder. HPLC analysis wason a C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.8 minutes and product 10.9minutes with >99% purity at 370 nm. ¹H-NHR (CD₃CN): δ (ppm) 1.2 (24H,bm, CH₃); 1.7 (8H, m, CH₂); 2.4 (12H, s, CH₃); 2.5 (12H, s, CH₃); 2.8(˜12H, bm, CH₂); 2.9 (˜8H, bm, CH₂); 3.1 (8H, m, CH₂); 3.6 (˜1800H, bs,PEG backbone); 3.9 (8H, s, CH₂); 4.0 (8H, bm, CH₂); 4.2 (8H, t, CH₂);7.0 (8H, m, Ar); 7.1 (4H, bm, NH); 7.5 (4H, m, Ar); 7.6 (˜4H, s, CH);9.1 (4H, s, NH); 12.7 (˜4H, s, NH); data above ˜12.8 ppm not available.Substitution 87% by NMR analysis.

Example 3 Synthesis of Hydroxyethylpiperazine Linked PEG-SunitinibConjugates

Synthesis of (Z)-(tert-butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate)2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1c)

Synthesis was conducted as described in Method A substituting tert-butyl4-(2-hydroxyethyl)piperazine-1-carboxylate (Compound c) (3.8 g, 17 mmol)in anhydrous tetrahydrofuran (12 mL) and anhydrous acetonitrile (8 mL)to give a orange suspension. Purified product yield was 0.2 g of yellowpowder. HPLC analysis was on a C18 silica column applying anacetonitrile gradient with 0.1% TFA; retention times observed weresunitinib 3.4 minutes and product 4.7 minutes with 99% purity at 370 nm.Analysis by LC-MS ([C₃₄H₄₈FN₆O₆]⁺ expected M+H=655.36. found M+H=655.3).¹H-NHR (CDCl₃): δ (ppm) 1.0 (6H, t, CH₃); 1.4 (9H, s, CH₃); 2.3 (4H, m,CH₂); 2.4 (3H, s, CH₃); 2.5 (3H, s, CH₃); 2.6 (4H, m, CH₂); 2.8 (2H, m,CH₂); 3.3 (4H, min, CH₂); 3.9 (2H, m, CH₂); 4.2 (2H, m, CH₂); 6.8 (1H,m, Ar); 6.9 (1H, m, Ar); 7.2 (1H, dd, Ar); 7.4 (1H, s, CH); 7.6 (˜1H, s,NH); data above ˜12.8 ppm not available.

Synthesis of (Z)-2-(piperazin-1-yl)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamatetrihydrochloride (Compound 2c)

Synthesis was conducted as described in Method B substituting Compound1c (0.19 g, 0.29 mmol). Crude yield ˜0.2 g orange solid. HPLC analysiswas on a C18 silica column applying an acetonitrile gradient with 0.1%TFA; retention times observed were sunitinib 3.5 minutes and product 2.4minutes with 97% purity at 370 nm. Analysis by LC-MS ([C₂₉H₄₀FN₆O₄,]⁺expected M+H=555.31. found M+H=555.3). ¹H-NHR (CD₃OD): δ (ppm) 1.5 (6H,s, CH₃); 2.4 (3H, s, CH₃); 2.5 (3H, s, CH₃); 3.2 (2H, m, CH₂); 3.4 (4H,m, CH₂); 3.5 (2H, m, CH₂); 4.2 (2H, m, CH₂); 4.4 (2H, bm, CH₂); 6.9-7.0(2H, m, Ar); 7.5 (1H, dd, Ar); 7.7 (1H, s, CH); data above ˜12.8 ppm notavailable.

Synthesis of (Z)-2-(4-(3-(mPEG 20,000)propanoyl)piperazin-1-yl)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 3c)

Synthesis was conducted as described in Method C substituting Compound2c (5.6 mg, 0.008 mmol). Yield 114 mg yellow powder.

Synthesis of (Z)-2-(4-(2-(4-armPEG 20,000)acetyl)piperazin-1-yl)ethyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4c)

Synthesis was conducted as described in Method D substituting Compound2c (0.17 g, 0.24 mmol). Yield was 1.0 g yellow powder. HPLC analysis wason a C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.5 minutes and product 11.0minutes with >99% purity at 370 nm. ¹H-NHR (CDCl₃): δ (ppm) 1.4 (24H,bs, CH₃); 2.2 (8H, s, CH₂); 2.3 (8H, s, CH₂); 2.3 (12H, s, CH₃); 2.4(12H, s, CH₃); 3.3 (8H, s, CH₂); 3.4 (˜8H, s, CH₂); 3.6 (˜1800H, bm, PEGbackbone); 4.1 (8H, s, CH₂); 4.2 (˜8H, bm, CH₂); 6.9 (8H, m, Ar); 7.2(4H, m, Ar); 7.3 (4H, s, CH); 8.8 (4H, s, NH); data above ˜12.8 ppm notavailable. Substitution 88% by NMR analysis.

Example 4 Synthesis of 4-Hydroxymethylpiperidine Linked PEG-SunitinibConjugates

Synthesis of (Z)-tert-butyl4-(((2-(diethylamino)ethyl)(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamoyloxy)methyl)piperidine-1-carboxylate(Compound 1d)

Synthesis was conducted as described in Method A substituting tert-butyl4-(hydroxymethyl)piperidine-1-carboxylate (Compound d) (2.1 g, 9.8 mmol)in anhydrous tetrahydrofuran (2.5 mL) and anhydrous acetonitrile (1 mL)to give a orange suspension. Purified product yield was 83 mg of yellowpowder. HPLC analysis was on a C18 silica column applying anacetonitrile gradient with 0.1% TFA; retention times observed weresunitinib 3.4 minutes and product 10.1 minutes with 97% purity at 370nm. Analysis by LC-MS ([C₃₄H₄₇FN₅O₆]⁺ expected M+H=640.35. foundM+H=640.3). ¹H-NHR (CD₃CN): δ (ppm) 0.8 (2H, bm, CH₂); 1.0 (6H, t, CH₃);1.3 (9H, s, CH₃); 1.5 (1H, bm, CH); 2.3 (3H, s, CH₃); 2.4 (3H, s, CH₃);2.4 (2H, bm, CH₂); 2.5 (4H, m, CH₂); 2.7 (2H, m, CH₂); 3.8 (6H, m, CH₂);6.8 (2H, m, Ar); 7.4 (1H, m, Ar); 7.5 (1H, s, CH); 8.8 (1H, s, NH); dataabove ˜12.8 ppm not available.

Synthesis of (Z)-(1-(3-(mPEG 20,000)propanoyl)piperidin-4-yl)methyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 2d)

Synthesis was conducted as described in Method B substituting Compound1d (80 mg, 0.13 mmol). Crude yield ˜80 mg orange solid. HPLC analysiswas on a C18 silica column applying an acetonitrile gradient with 0.1%TFA; retention times observed were sunitinib 3.5 minutes and product 2.1minutes with 97% purity at 370 nm. Analysis by LC-MS ([C₂₉H₃₉FN₅O₄,]⁺expected M+H=540.30. found M+H=540.3). ¹H-NHR (d₆-DMSO): δ (ppm) 1.2(8H, m, CH₃, CH₂); 1.5 (2H, m, CH₂); 1.7 (1H, bm, CH); 2.3 (3H, s, CH₃);2.4 (3H, s, CH₃); 2.7 (2H, m, CH₂); 3.1 (2H, m, CH₂); 3.2 (4H, m, CH₂);3.3 (2H, m, CH₂); 4.0 (2H, m, CH₂); 4.1 (2H, m, CH₂); 6.9 (1H, m, Ar);7.0 (1H, m, Ar); 7.8 (1H, s, CH); 7.8 (1H, dd, Ar); 8.5 (1H, bs, NH);8.6 (1H, bs, NH); 10.2 (1H, bs, NH); 11.0 (1H, s, NH); 13.9 (1H, s, NH);data above ˜12.8 ppm not available.

Synthesis of (Z)-(1-(3-(mPEG 20,000)propanoyl)piperidin-4-yl)methyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 3d)

Synthesis was conducted as described in Method C substituting Compound2d (16 mg, 0.025 mmol). Yield 0.3 g yellow powder. HPLC analysis was ona C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.5 minutes and product 10.4minutes with >99% purity at 370 nm. ¹H-NHR (CD₃CN): δ (ppm) 0.8 (2H, bm,CH₂); 1.0 (6H, t, CH₃); 1.2-1.4 (˜2H, bm, CH₂); 1.6 (1H, bm, CH); 2.3(3H, s, CH₃); 2.4 (3H, s, CH₃); 2.5 (9H, s, CH₃); 2.7 (2H, t, CH₂); 2.8(1H, t, CH₂); 3.3 (3H, s, OCH₃); 3.6 (˜1800H, bs, PEG backbone); 3.8-3.9(˜5H, bm, CH₂); 4.3 (1H, m, CH₂); 6.9 (2H, m, Ar); 7.4 (1H, dd, Ar); 7.6(1H, s, CH); 9.1 (˜1H, bs, NH); data above ˜12.8 ppm not available.Substitution by NMR 89%.

Synthesis of (Z)-(1-(2-(4-armPEG 20,000)acetyl)piperidin-4-yl)methyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4d)

Synthesis was conducted as described in Method D substituting Compound2d (72 mg, 0.11 mmol). Yield was 0.45 g yellow powder. HPLC analysis wason a C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.5 minutes and product 10.8minutes with >98% purity at 370 nm. ¹H-NHR (CDCl₃): δ (ppm) 0.8 (4H, bm,CH₂); 1.0 (˜4H, bm, CH₂); 1.2 (˜8H, bm, CH₂); 1.4 (˜24H, bs, CH₃); 1.5(˜8H, bm, CH₂); 1.7 (˜8H, bm, CH₂); 2.3 (˜12H, s, CH₃); 2.4 (˜12H, s,CH₃); 2.8 (˜4H, bm, CH); 3.1 (˜16H, bm, CH₂); 3.6 (˜1800H, bs, PEGbackbone); 4.1 (8H, s, CH₂); 4.1 (˜24H, bm, CH₂); 4.4 (˜8H, d, CH₂); 6.9(8H, m, Ar); 7.2 (4H, d, Ar); 7.3 (4H, s, CH); 9.0 (˜4H, bs, NH); dataabove ˜12.8 ppm not available. Substitution 94% by NMR analysis. ¹H-NHR(CD₃CN): δ (ppm) 0.8 (8H, bm, CH₂); 1.0 (˜24H, bs, CH₂); 1.3 (˜8H, bin,CH₂); 1.6 (˜4H, bm, CH₂); 2.3 (˜12H, s, CH₃); 2.4 (˜12H, s, CH₃); 2.6(˜12H, bm, CH); 2.8 (˜12H, bm, CH); 3.5 (˜1800H, bs, PEG backbone); 3.9(˜16H, m, CH₂); 4.0 (8H, s, CH₂); 4.4 (˜4H, d, CH); 6.9 (8H, d, Ar); 7.4(4H, d, Ar); 7.5 (4H, s, CH); 9.0 (˜4H, s, NH [oxindole]); 13.7 (˜4H, s,NH [pyrrole]).

Example 5 Synthesis of Glycerol Linked PEG-Sunitinib Conjugates

Synthesis of (Z)-2,3-dihydroxypropyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1e)

Synthesis was conducted as described in Method A substituting glycerol(Compound e) (84 mg, 0.91 mmol) in anhydrous tetrahydrofuran (0.2 mL)and anhydrous acetonitrile (0.2 mL) to give a orange suspension. Crudeproduct was an orange semi-solid. HPLC analysis was on a C18 silicacolumn applying an acetonitrile gradient with 0.1% TFA; retention timesobserved were sunitinib 3.6 minutes and product 3.3 minutes with 54%purity at 370 nm. Analysis by LC-MS ([C₂₆H₃₄FN₄O₆]⁺ expected M+H=517.25.found M+H=517.2).

Example 6 Synthesis of Amino-Hexanol Linked PEG-Sunitinib Conjugates

Synthesis of (Z)-(tert-butyl 6-hydroxyhexylcarbamate)2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1g)

Synthesis was conducted as described in Method A substituting tert-butyl6-hydroxyhexylcarbamate (Compound g) (1.1 g, 5.0 mmol) to give a orangesuspension. Purified product yield was 13 mg of yellow powder. HPLCanalysis was on a C18 silica column applying an acetonitrile gradientwith 0.1% TFA; retention times observed were sunitinib 3.4 minutes andproduct 10.9 minutes with 95% purity at 370 nm. Analysis by LC-MS([C₃₄H₄₉FN₅O₆]⁺ expected M+H=642.37. found M+H=642.4).

Synthesis of (Z)-6-aminohexyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamatetrihydrochloride (Compound 2g)

Synthesis was conducted as described in Method B substituting Compound1g (13 mg, 0.02 mmol). Crude yield ˜14 mg orange solid. HPLC analysiswas on a C18 silica column applying an acetonitrile gradient with 0.1%TFA; retention times observed were sunitinib 3.5 minutes and product 2.6minutes with 96% purity at 370 nm. Analysis by LC-MS ([C₂₉H₄₁FN₅O₄,]⁺expected M+H=542.31. found M+H=542.3).

Synthesis of (Z)-6-(2-(4-armPEG 20,000)acetamido)hexyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4g)

Synthesis was conducted as described in Method D substituting Compound2g (13 mg, 0.02 mmol). Yield was 42 mg yellow powder. HPLC analysis wason a C18 silica column applying an acetonitrile gradient with 0.1% TFA;retention times observed were sunitinib 3.4 minutes and product 10.8minutes with >99% purity at 370 nm. ¹H-NHR (CD₃CN): δ (ppm) 1.1 (˜28H,bm, CH₃, CH₂); 1.1 (˜8H, m, CH₂); 1.3-1.4 (˜20H, m, CH₂); 2.3 (12H, s,CH₃); 2.4 (12H, s, CH₃); 2.6 (16H, bm, CH₂); 2.7 (˜8H, bm, CH₂); 3.0(˜8H, bm, CH₂); 3.5 (˜1800H, bs, PEG backbone); 3.8 (8H, s, CH₂); 3.9(8H, bm, CH₂); 4.0 (8H, bm, CH₂); 6.9 (˜8H, m, Ar); 7.0 (˜4H, bm, NH);7.4 (4H, m, Ar); 7.6 (4H, s, CH); 9.1 (˜4H, s, NH); 12.7 (˜4H, s, NH);data above ˜12.8 ppm not available. Substitution 91% by NMR analysis.¹H-NHR (CD₃CN): δ (ppm) 1.0 (˜32H, bm, CH₂); 1.1 (˜8H, bs, CH₂); 1.3-1.4(˜20H, bm, CH₂); 2.3 (˜12H, s, CH₃); 2.4 (˜12H, s, CH₃); 2.6 (˜12H, bm,CH); 2.7 (˜8H, bm, CH); 3.0 (˜8H, bm, CH); 3.5 (˜1800H, bs, PEGbackbone); 3.9 (˜8H, s, CH₂); 4.0 (8H, bm, CH₂); 4.2 (˜8H, d, CH); 6.9(˜8H, d, Ar); 7.0 (˜4H, NH [amide-PEG]); 7.4 (˜4H, d, Ar); 7.6 (˜4H, s,CH); 9.0 (˜4H, s, NH [oxindole]); 13.8 (˜4H, s, NH [pyrrole]).

Example 7 Synthesis of Amino-Butanol Linked PEG-Sunitinib Conjugates

Synthesis of (Z)-(tert-butyl 4-hydroxybutylcarbamate)2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 1h)

Synthesis was conducted as described in Method A substituting tert-butyl4-hydroxybutylcarbamate (Compound h) (2.0 g, 10.7 mmol) in anhydroustetrahydrofuran (2 mL) to give a orange suspension. Purified productyield was 147 mg of yellow powder. HPLC analysis was on a C18 silicacolumn applying an acetonitrile gradient with 0.1% TFA; retention timesobserved were sunitinib 3.5 minutes and product 9.2 minutes with 97%purity at 370 nm. Analysis by LC-MS ([C₃₂H₄₅FN₅O₆]⁺ expected M+H=614.33.found M+H=614.3). ¹H-NHR (CD₃CN): δ (ppm) 1.0 (6H, t, CH₃); 1.2 (2H, m,CH₂); 1.3 (9H, s, CH₃); 2.3 (3H, s, CH₃); 2.4 (3H, s, CH₃); 2.5 (4H, m,CH₂); 2.7 (2H, m, CH₂); 3.0 (2H, m, CH₂); 3.9 (2H, m, CH₂); 4.1 (2H, m,CH₂); 5.1 (˜1H, bs, NH); 6.9 (2H, m, Ar); 7.4 (1H, dd, Ar); 7.5 (1H, s,CH); 8.8 (1H, bs, NH); data above ˜12.8 ppm not available.

Synthesis of (Z)-4-aminobutyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamatetrihydrochloride (Compound 2h)

Synthesis was conducted as described in Method B substituting Compound1h (0.13 mg, 0.21 mmol). Crude yield ˜0.13 g orange solid. HPLC analysiswas on a C18 silica column applying an acetonitrile gradient with 0.1%TFA; retention times observed were sunitinib 3.4 minutes and product 2.1minutes with 98% purity at 370 nm. Analysis by LC-MS ([C₂₇H₃₇FN₅O₄,]⁺expected M+H=514.28. found M+H=514.3). ¹H-NHR (CD₃OD): δ (ppm) 1.4 (6H,t, CH₃); 1.4 (2H, m, CH₂); 1.5 (2H, m, CH₂); 2.4 (3H, s, CH₃); 2.5 (2H,s, CH₃); 2.8 (2H, t, CH₂); 3.4 (2H, m, CH₂); 3.6 (2H, m, CH₂); 4.2 (2H,m, CH₂); 6.9 (2H, m, Ar); 7.5 (1H, dd, Ar); 7.6 (1H, s, CH); data above˜12.8 ppm not available.

Synthesis of (Z)-4-(2-(4-armPEG 20,000)acetamido)butyl2-(diethylamino)ethyl(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl)carbamate(Compound 4h)

Synthesis was conducted as described in Method D substituting Compound2h (125 mg, 0.20 mmol). Yield was 0.83 g yellow powder. HPLC analysiswas on a C18 silica column applying an acetonitrile gradient with 0.1%TFA; retention times observed were sunitinib 3.4 min and product 10.8min with 97% purity at 370 nm. ¹H-NHR (CD₃CN): δ (ppm) 1.1 (24H, bm,CH₃); 1.3 (8H, m, CH₂); 1.5 (8H, m, CH₂); 2.3 (12H, s, CH₃); 2.4 (12H,s, CH₃); 2.6-3.1 (16H, bm, CH₂); 3.0 (8H, m, CH₂); 3.5 (˜1800H, bs, PEGbackbone); 3.8 (8H, s, CH₂); 4.0 (16H, bm, CH₂); 6.9 (8H, m, Ar); 7.0(4H, bm, NH); 7.4 (4H, m, Ar); 7.5 (4H, s, CH); 9.0 (˜4H, s, NH); dataabove 12.8 ppm not available. Substitution 93% by NMR analysis.

Example 8 Alternate Synthesis of Amino-Propanol-Linked PEG-SunitinibConjugates

An alternate synthesis of amino-propanol-linked PEG-sunitinib conjugateswas conducted using an approach schematically represented below.

Synthesis of (Z)-3-(tert-butoxycarbonylamino)propyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 1i)

In a 50 mL round-bottom flask was dissolved tert-butyl3-hydroxypropylcarbamate (Compound b) (0.2 mL, 1.1 mmol) in anhydrousacetonitrile (0.8 mL). A suspension of di(1-benzotriazolyl)carbonate(Di-BTC) in 1,1,1-trichloroethane (˜67% Di-BTC by weight, 0.38 g, 0.87mmol) was added and followed by addition of anhydrous pyridine (0.28 mL,3.4 mmol). After one hour, the solvent was evaporated under reducedpressure. To the crude product (Compound i) was added a solution ofsunitinib (34 mg, 0.09 mmol) in warm anhydrous pyridine (2.2 mL).Anhydrous triethylamine (0.55 mL was added. After four days, the solventwas evaporated under reduced pressure. The crude red-orange product wasdissolved in methanol (0.4 mL) and was purified further on a BiotageFlash silica column with a DCM/MeOH gradient program. Product fractionswere combined and evaporated at reduced pressure. Purified product was ayellow powder. HPLC analysis was on a C18 silica column applying anacetonitrile gradient with 0.1% TFA; retention times observed weresunitinib 4.5 minutes and product 11.4 minutes with 95% purity at 370 nm(noted compound 1b with same HPLC gradient method retention time was10.2 min). Analysis by LC-MS ([C₃₁H₄₃FN₅O₆]⁺ expected M+H=600.32. foundM+H=600.3).

Synthesis of (Z)-3-aminopropyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylatetrihydrochloride (Compound 2i)

Synthesis was conducted as described in Method B substituting(Z)-3-(tert-butoxycarbonylamino)propyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 1i) (30 mg, 0.05 mmol). Crude yield ˜42 mg orange solid. HPLCanalysis was on a C18 silica column applying an acetonitrile gradientwith 0.1% TFA; retention times observed were sunitinib 4.3 minutes andproduct 2.7 minutes with 95% purity at 370 nm.

Synthesis of (Z)-3-(3-(mPEG 20,000)propanamido)propyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 3i)

Synthesis was conducted as described in Method C substituting(Z)-3-aminopropyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylatetrihydrochloride (Compound 2i) (21 mg, 0.03 mmol). Yield ˜150 mg yellowpowder. HPLC analysis was on a C18 silica column applying anacetonitrile gradient with 0.1% TFA; retention times observed weresunitinib ˜3.7 minutes and product 11.1 minutes with 99% purity at 370nm.

Example 9 Synthesis of Ethylene Glycol Linked PEG-Sunitinib Conjugates

Synthesis of (Z)-2-(mPEG 5,000)ethyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 5j)

In a 50 mL round-bottom flask was dissolved sunitinib (0.11 g, 0.27mmol) in warm anhydrous pyridine (2 mL). After the sunitinib solutioncooled to room temperature, mPEG_(5k)-carbamoyl-methylmidizole triflate(0.45 g, 0.09 mmol) was added. After 18 hours, the crude product wasprecipitated by the addition of diethyl ether and collected in a filterfunnel. The isolated crude product was dissolved in warm anhydrous IPAand slowly cooled to room temperature forming precipitate. The resultingslurry was filtered and washed with additional anhydrous IPA. Residualsolvent was evaporated at reduced pressure. HPLC analysis was on a C18silica column applying an acetonitrile gradient with 0.1% TFA; retentiontimes observed were sunitinib 5.6 minutes and product 6.7 minutes with93% purity at 370 nm.

Alternate synthesis of (Z)-2-(mPEG 5,000)ethyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 5j)

In a 50 mL round-bottom flask was dissolved sunitinib (0.34 mg, 0.09mmol) in anhydrous 1,4-dioxane (1.5 mL) and triethylamine (0.12 mL, 0.9mmol) at ˜50° C. To the sunitinib solution was added mPEG_(5k)-BTC (0.45g, 0.09 mmol). After ˜1.5 days, the solvent was evaporated under reducedpressure to a thick oil. The crude product was dissolved in warmanhydrous IPA and slowly cooled to room temperature forming precipitate.The resulting slurry was filtered and washed with additional anhydrousIPA. Residual solvent was evaporated at reduced pressure.

Example 10

Synthesis of Ethylene Glycol Linked PEG-Semaxanib Conjugates

Synthesis of(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carbonylchloride (Compound 6)

In a 50 mL round-bottomed flask was suspended(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2-one(semaxanib) (0.11 g, 0.47 mmol) in anhydrous THF (5 mL). The suspensionwas transferred to the triphosgene reaction in a second flask.

(Caution: To prevent release of toxic phosgene gas from either thereaction apparatus or rotary evaporator, the equipment setups weresparged through a sodium hydroxide scrub solution via an over pressureor exhaust port.) In a separate 50 mL round-bottomed flask was addedtriphosgene (1.6 g, 5.4 mmol) in anhydrous THF (40 mL) to give acolorless solution. Triethylamine (1.1 mL, 7.8 mmol) was added. Afterten minutes, a semaxanib solution was transferred into this triphosgenesolution. After approximately one hour, the reaction flask was placed onice and cold 4M HCl solution (30 mL) was added to the flask. The crudeproduct suspension was stirred for ten minutes, filtered and washed withcold 4M HCl solution (30 mL). The crude product was then placed underhigh vacuum for 18 hours in the presence of P₂O₅. Crude yield (Compound6) was 0.12 g of a red solid. HPLC analysis was on a C18 silica columnapplying an acetonitrile gradient with 0.1% TFA; retention timesobserved were semaxanib 6.2 minutes and carbamoyl chloride product 7.4minutes with ≧33% substitution at 280 nm. The carbamoyl chloride productwas further characterized by reaction with excessn-butylamine(Z)—N-butyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxamideand analyzed by HPLC. HPLC analysis was on a C18 silica column applyingan acetonitrile gradient with 0.1% TFA; retention times observed weresemaxanib 6.2 minutes and butylamine derivative 7.7 min with 81%substitution at 280 nm.

Synthesis of (Z)-(mPEG 20,000)3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 6k)

In a 50 mL flask was dissolved mPEG-OH 20K (0.5 g, 0.025 mmol) inanhydrous toluene. The solvent was evaporated under reduced pressure.The polymer was dissolved in anhydrous DCM (0.5 mL) and pyridine (0.02mL, 0.23 mmol). To the polymer solution was added a suspension of crude(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carbonylchloride (Compound 6) (23 mg, 0.08 mmol) in anhydrous THF (2.5 mL).After one day, additional Compound 6 (28 mg, 0.1 mmol) was added. After˜3 days, the solvent was evaporated under reduced pressure to a thickoil. The crude product was dissolved in warm anhydrous IPA and slowlycooled to room temperature forming precipitate. The resulting slurry wasfiltered and washed with additional anhydrous IPA. Residual solvent wasevaporated at reduced pressure. Yield was ˜0.45 g of a solid powder.HPLC analysis was on a C18 silica column applying an acetonitrilegradient with 0.1% TFA; retention times observed were semaxanib 4.7minutes and product 4.9 minutes with 96% purity at 280 nm and 59% purityby ELSD. ¹H-NHR (d₆-DMSO): δ (ppm) 2.3 (˜3H, s, CH₃); 2.4 (˜3H, s, CH₃);3.2 (˜3H, s, CH₃); 3.6 (˜1800H, bs, PEG backbone); 4.5 (˜2H, s, CH₂);4.6 (<1H, m, OH); 6.1 (˜1H, s, CH); 7.2 (˜2H, m, Ar); 7.7 (˜1H, s, CH);7.8 (˜1H, m, Ar); 7.9 (˜1H, m, Ar); 12.6 (˜1H, s, NH); data above ˜12.8ppm not available.

Example 11 Half-Lives of Conjugates

The half-lives observed for several sunitinib conjugates of theinvention were determined in buffer and plasma according to thedescriptions in “Method E” provided in the Experimental. The data isprovided in Table 3.

TABLE 3 Release Half-lives Observed for Sunitinib Conjugates in Bufferand Plasma Conjugate Name Rat Plasma Dog Plasma Phosphate pH 7.5Phosphate pH 6.8 Compound 4a  ~8-12 h    ~17 h 2.3 d 10 d Compound 4b~30-52 h ~37-51 h 2.3 d Compound 3b ~11-16 h ~17-21 h 2.5 d Compound 3i (~7-13 h) (~25-36 h) 9.3 d Compound 4c ~19-33 h ~29-36 h 1.1 d 4.4 dCompound 4d ~39-61 h ~57-71 h 3.8 d 18 d Compound 1e ~3 min ~10 minCompound 4g ~65-81 h ~102-165 h  5.5 d 21 d Compound 4h ~47-57 h ~44-69h 4.3 d 17 d Compound 5j 6.6 d Compound 6k ~>3 d NOTE: Italic textindicates poor data fit to first order plot.

Example 12 In Vivo Release Kinetics and Tumor Accumulation

Eight to twelve week old female NCr nu/nu mice with were injectedsubcutaneously with 5×10⁶ HCT116 colorectal cancer cells in 0% Matrigelin a flank. Upon reaching a tumor size of 500 mm³, mice were treatedwith either 40 mg/kg sunitinib or 40 mg/kg sunitinib equivalent ofCompounds 4a, 4d and 4g (IV via tail vein). Blood and tumor samples werecollected at 6, 12, 24, 48, 72, 120 and 168 hours post dose to determineplasma and tumor sunitinib concentrations.

Data is provided in FIG. 3, where mean the plasma concentration ofsunitinib following administration of a compound of interest is providedfor a series of time points. Turning to FIG. 4, tumor concentration ofsunitinib following administration of a compound of interest is providedfor a series of time points.

Example 13 Alternate Synthesis of Amino-Diethyleneglycol-LinkedPEG-Sunitinib Conjugates

An alternate synthesis of amino-diethyleneglycol-linked PEG-sunitinibconjugates was conducted using an approach schematically representedbelow.

Synthesis of (Z)-2-(2-(tert-butoxycarbonylamino)ethoxy)ethyl3-((4-(2-(diethylamino)ethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 1m)

In a 50 mL round-bottom flask was dissolved tert-butyl3-hydroxypropylcarbamate (Compound b) (1.35 g, 6.6 mmol) in anhydrousacetonitrile (30 mL). A suspension of di(1-benzotriazolyl)carbonate(Di-BTC) in 1,1,1-trichloroethane (˜67% Di-BTC by weight, 2.23 g, 5.0mmol) was added and followed by addition of anhydrous pyridine (1.64 mL,20.3 mmol). After one hour, the solvent was evaporated under reducedpressure. To the crude product (Compound m) was added a solution ofsunitinib (198.5 mg, 0.5 mmol) in warm anhydrous pyridine (13 mL).Anhydrous triethylamine (3 mL) was added. After 5 days, hexanes (170 mL)was added to the reaction mixture. The hexanes layer was removed leavingcrude product as an orange oil. Additional hexanes (150 mL) was added,mixed and then hexanes layer was removed. The crude red-orange productwas dissolved in DCM and was purified further on a Biotage Flash silicacolumn with a DCM/MeOH gradient program. Product fractions were combinedand evaporated at reduced pressure. Purified product was an orange film.HPLC analysis was on a C18 silica column applying an acetonitrilegradient with 0.1% TFA; retention times observed were sunitinib 4.3minutes and product 10.1 minutes with 96% purity at 370 nm. Analysis byLC-MS ([C₃₁H₄₃FN₅O₆]⁺ expected M+H=630.33. found M+H=630.2). HPLC-MS onC18 silica column applying an acetonitrile gradient with 0.1% formicacid; Co-injection of Compound 1a and Compound 1m: retention times forsunitinib 3.7 minutes (M+H=399.1), Compound 1a 9.0 minutes (M+H=630.2),Compound 1m 9.7 minutes (M+H=630.2). ¹H-NMR (d₆-DMSO): δ (ppm) 1.1 (6H,t, CH₃); 1.3 (9H, s, CH₃); 2.4 (3H, s, CH₃); 2.5 (3H, s, CH₃); 2.8 (˜6H,bm, CH₂); 3.1 (2H, m, CH₂); 3.4 (2H, bm, CH₂); 3.5 (2H, t, CH₂); 3.8(2H, m, CH₂); 4.5 (2H, m, CH₂) 6.8 (˜1H, t, NH [Boc]); 7.0 (1H, m, Ar);7.6 (1H, s, CH); 7.7 (1H, m, Ar); 7.8 (1H, m, Ar); 7.8 (1H, bs, NH[pyrrole amide]); 12.7 (1H, s, NH [pyrrole]). ¹³C-NMR (d₆-DMSO): δ (ppm)10.7 (3C, CH₂, CH₃); 13.4 (CH₃); 28.1 (3C, CH₃); ˜39.5 (CH₂); 46.6 (2C,CH₂); 51.0 (2C, CH₂); 65.8 (CH₂); 67.6 (CH₂); 69.2 (CH₂); 77.6 (C);105.1, 105.3 (d, Ar); 111.0 (Ar); 112.4, 112.6 (d, Ar); 115.6, 115.7 (d,Ar); 121.2 (pyrrole); 125.7, 125.8 (CH, pyrrole); 127.2, 127.3 (d, C);131.2 (Ar); 133.0 (pyrrole); 138.8 (pyrrole); 149.9 (C(O)); 155.6(C(O)); 158.6, 160.4 (d, Ar); 164.4 (C(O)); 166.3 (C(O)).Characterization was supported by 2D-NMR experiments including:¹H-¹H-COSY, ¹H-¹³C-HSQC, and ¹H-¹³C-HMBC. Exchangeable protons wereevaluated by the addition of H₂O to the NMR sample in d₆-DMSO whichdemonstrated loss of integration for δ (ppm) 7.8 (NH [pyrrole amide]),significantly diminished integration for δ (ppm) 12.7 (NH [pyrrole]) andnearly similar integration for δ (ppm) 6.8 (NH [Boc]).

What is claimed is:
 1. A compound encompassed within the formula

and pharmaceutically acceptable salts thereof, wherein each n is aninteger from about 100 to about
 2270. 2. A composition comprising (i) acompound, of claim 1, and (ii) a pharmaceutically acceptable excipient.3. The compound of claim 1, wherein each n is an integer from about 136to about
 2050. 4. The compound of claim 3, wherein each n is an integerfrom about 225 to about 1930.